Method for the early diagnosis of cancer

ABSTRACT

The present invention relates to a method for the early diagnosis of cancer in a subject, which is based on determination of the relative fraction of microorganisms derived from the feces of the sublect, as compared to the total count of microorganisms in the same of corresponding sample. This relation has been found to be indicative of ihe presence or absence of cancer in said subject.  
     After isolating at least one of the microorganisms from the fecal sample to form a so-called diagnostic sample, and incubating, for a sufficient time, the diagnostic sample with cancer cells. the microorganism being in an amount corresponding to its relative fraction in the original fecal sample, the cancerolytic activity of the microoganism/s is indicative to the presence or absence of cancer cells in the subject. The cancerolytic activity is expressed by terms of a tumor cell nccrosis index (TCNI).  
     Further, the method of the invention is based on determinig level of expression or level of activity of L-PAR II in a fecal-derived micoorganism/s sample, the level determined being also indicative of the presence or absence of cancer cells in the subject from which the fecal sample was derived.

FIELD OF THE INVENTION

[0001] This invention relates to methods for the early diagnosis cancer.

BACKGROUND OF THE INVENTION

[0002] It is generally agreed at early diagnosis of cancer is almostalways a prerequisite of sucessful treatment. For example, the WHOExpert Committee's Report on Early Detection of Cancer (1969) statedthat over half of cancer patients could have been cured if the diseasehad been detected at an early stage and treated soon after detection. Inview of the widespread incidence of the disease, mass screeningtechniques would evidently be of great value, such as is available, forexample, at least in developed countries, in the field of tuberculosisof the chest by means of mass X-ray examination.

[0003] Among previous proposals for the diagnosis of cancer may bemotioned the following. In U.S. Pat No. 3,476,514, there was described amethod of detecting cancer cells by staining test cells withacriflavine-HCl solution, determining indirectly the dye absorbed by thetest cells and comparing with a contol. JP. 54,143,528 proposed a methodfor diagnosing malignant tumors which utilized an injectable compositioncontaining an endotoxin extracted from cultured bacteria. In GB 1587244,there was described inter alia, the use in a serum agglutination test onthe sera of patients, for the detetion of neoplasm, of an antigenproduced by a species of the genus Streptococcus.

[0004] Bodily health is known to be affected by the nature of theintestinal flora which apparently influences, for example, metabolicprocesses and both local and general body immune response. It has alsobeen known for some time that certain of the intestinal flora bacteriaof normal humans have onolytic activity, and that there exists arelationship between intestinal microfloral composition and cancermorbidity, see e.g., Oleynik, S. F. and Panchishina, M. V., “AboutColiflora and Cancerolycity aod Carcinogenicity of the intestine”,Vrachebnoye-delo, 5:13-17 (1968).

[0005] There are several publications relating to the use of fecalsamples for the detection of cancer. For example, EP 685739 described amethod for the diagnosis of colorectal cancer comprising reactionanti-DAF antibodies with a suparnatant of a fecal sample thereby formingan antigen-antibody complex and measuring the amount of complex formed.Another publication is U.S. Pat No. 5,952,200 which describes a test fordiagnosing the presence of cancerous cells in samples of human tissue,fluids or semi-fluids such as feces, by the detection of transcripts forstromelysin0-3 in the tissue cells.

[0006] JP 6258324 describes a method for the diagosis ofdigestive-system cancer disease by using an antibody selective tovariation-type protein of the cancer-suppressing gene P53, while U.S.Pat. No. 5,455,160 describes a method for determination of fecalcalprotectin as a parameter for monitoring Inflammatory Bowel Diseaseand gastrointestinal cancer.

[0007] Finally, U.S. Pat. No. 5,344,762 describes a method for earlydiagnosis of cancer by incubating in vitro a human fecal sampleincluding bacteria with a standard culture of a known number of cancercells. After a time period the number of cancer cells interacted andnon-interacted with the bacteria is counted and the results are used fordetermination of a disease state.

SUMMARY OF THE INVENTION

[0008] It has now been found that healithy individuals and cancerpatients show striking differences in their fecal microflora. Inparticular, healthy individuals were shown to present a higherpercentage of Esherichia coli (E. coli) with concerolytic activity ascompared to the microflora of cancer patient.

[0009] Thus, according to a first of its aspects, the present inventionprovides a method for the early diagnosis of cancer in a subject, themethod comprising the steps of:

[0010] i) providing a fecal sample from said subject;

[0011] ii) treating said sample to obtain a feces-derived microorganismssample;

[0012] iii) identifying in the microorganism sample one or more types ofmicroorganisms contained therein and;

[0013] iv) determining for said one or more microorganisms its relativefraction from the total count of microorganism in said sample or in acorresponding sample, the relative fractions being indicative of thepresence or absence of cancer is said subject.

[0014] The term “early diagnosis of cancer” as used hereininterchangeably with the terms “early detection of cancer”, “cancerscreening” or “confirmation of cancer” is intended to convey suchdiagnosis, whether or not the cancer has reached the stage in which itis detectable by other methods presently availbale to the clinician.

[0015] The present invention also provides, according to a second of itsaspects, a method for an early diagnosis of cancer in a subjectcomprising the steps:

[0016] i) providing a fecal sample from said subject;

[0017] ii) treating said fecal sample to obtain a feces-derivedmicroorganism sample therefrom;

[0018] iii) identifying in the bacteria sample one or more types ofmicroorganisms;

[0019] iv) determining for each of said microorganisms its relativefraction from the total count of microorganisms in said sample or in acorresponding sample;

[0020] v) isolating one or more microorganisms from said sample forwhich their relative fraction was determined;

[0021] vi) preparing a diagnostic sample containing one or more of theisolated microorganisms, the fraction of the microorganisms in saiddiagonstic sample coorespding to the relative fraction thereof in thefecal sample, as determined in step (iv); and

[0022] vii) interacting said diagnostic sample with cancer cells for atime period sufficient to detect lysis of said cancer cells by themicroorganism in said diagnostic sample, thereby determining for saidfecal sample a tumor cell necrosis index (TCNI).

[0023] As indicated above, the diagnostic sample may contain one or moremicroorganisms. When containing a single microorganism, the latter isdiluted in the suitable medium to a concentration corresponding to itsrelative concentration in the original fecal sample. However, when usingtwo or more isolated microorganisms they are re-mixed to form adispersion of microorganisms in which the fraction of eachmicrooraganism corresponds to its relative fraction in the orginal fecalsample. The formation of the diagnostic sample may be referred toherein, at times, as the re-mixing step.

[0024] An important feature of this aspect of the ivention involvesremoval of contaminations from the sample before preparing thediagnostic sample from the isolated microorganism according to theirrelative fraction in the original fecal sample. Therefore, treating thefecal sample to obtain a feces-derived microorganism sample thereformincludes, removal of undesired contamination from the fecal sample toobtain an uncontaminated feces-derived microorganism sample. It has beenfound that the presence of contamination form the feces-derivedmicroorganism samples, results in deviation in the value obtained forthe TNCI.

[0025] Finally and in accordance with a third aspect of the presentinvention, there is provided a method for an early diagnosis of cancerin a subject comprising the steps:

[0026] i) providing a fecal sample from said subject;

[0027] ii) treating said sample to obtain a feces-derived microorganismsample;

[0028] iii) identifying in the microorganism sample one or more types ofmicroorganisms contained the and isolating at least one microorganismcapable of expressing in a healthy subject L-asparaginase II (L-PAR II);and

[0029] iv) determining level of expression of L-PAR II by the isolatedmicroorganism or level of asparinase hydrolysis by said L-PAR II, saidlevel is indicative of the presence or absence of cancer cells in saidsubject.

[0030] As will he shown in the following non-limiting examples, thelevel of expression L-PAR II or level of asparinase hydrolysis by theenzyme indicates the probability of an individual to have cancer. Inparticular, low levels of expression of L-PAR II as well as low levelsof asparaginase hydrolysis may suggest the high probability of a subjectto have cancer.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 is a schematic illustration of the method of thie presentinvention.

[0032] FIGS. 2A-2B are pictures obtained by microscope of cancer cellstreated wih the diagnostic sample of th present invention, thediagnostic samples derived either from a healthy subject (FIG. 2A) orform a cancer patient (FIG. 2B).

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is based on the finding that there is acorrelation between the relative fraction of microorganisms in the fecesand the probability of a subject to have cancer. In addition, whenincubating standard cancer cells with specific microorganisms isolatedfrom feces of a subject susceptible of carrying cancer cells, themicroorganisms being mixed in a relation corresponding to that presentin the feces of the individual, their capability to lyse the cancercells was found to be indicative of the absence or presence of cancercells in the subject and of tie level of disease in said subject.

[0034]FIG. 1 provides a schematic illustration of the different aspectsof the method of the present invention.

[0035] Thus, according to a first of its aspects, the present inventionprovides a method for the early diagnosis of cancer in a subject. Themethod comprises the steps of (i) providing a feacal sample (step 10)from said subject; (ii) treating said sample to obtain a feces-derivedmicroorganisms sample, for example, by removal therefrom undesiredcontamination (step 12), such as aggressive microorganism which may leadto deviation in the screening; (iii) identifying in the microorganismssample one or more microorganisms contained therein (step 14); and (iv)finally determining for each of said microorganisms its relativefraction from the total count of microorganisms in said sample or in acorresponding sample (i.e. a second sample from the same subject, beingtreated in the same manner as the tested sample) (step 16), the relativefractions being indicative of the prescence or absence of cancer in saidsubject (step 18)

[0036] The subject to be diagnosed for cancer is preferably a mammaliananimal and more preferably a human.

[0037] According to one embodiment, any type of the various types ofmalignant neoplasms derived from epithelial tissue (i.e. carcinomas) maybe detected by the method of the present invention. As will beappreciated by those versed in the art of medicine, such neoplasms occurfrequently in the skin, large intesine, lung, prostate gland and breastsamong other organs. Therefore, according to one preferred embodiment themethod of the present invention may be utilized for the early detectionof any one of the above carcinoma.

[0038] The relative fraction in the fecal sample of each type of themicroorganisms is determined by calculating the percentage of saidmicroorganism from the total count of microorganisms in the same fecesderived sample. In order to determine the same, microorganisms arepreferably first treated, e.g., to remove undesired contaminations,followed by isolation of the microorganisms by any conventionalisolating technique. For example, the microorganisms may be isolated bycolonies formation on selective culture mediums, as will furtherdetailed hereinafter.

[0039] According to one embodiment the microorganisms employed by thescreening method of the present invention are bacteria. One group offeces-derived bacteria includes, without being limited thereto,gram-negative anaerobic bacteria. Examples of such gram negativebacteria that typically inhabit the intestinal tracts Escherichia,Salmonella, Shigella, klebsiella, Yersmisa, Enterobacter, IIemophilus,Gardnerella and Pasteurella.

[0040] According to one preferred embodiment, the bacteria employed bythe method of the present invention are E. coli. these bacteria may beisolated from the feces-derived sample by the use, for example, ofculture medium selective for E. coli, such as MacConkey agar or m-Endoagar.

[0041] In addition, the bacteria may be a gram-positive bacteria.Examples of gram-positive bacteria include, inter alia, Staphylococcus,Enterococcus, Streptococcus, Luctococcus. According to one preferredembodiment the bacteria is Streptococcus bovis or Enterococcus sp.

[0042] The Enterococci coliform may be isolated from the fecal sample byculturing the feces-derived sample on a culture medium selective forEnterococcus. Non-limiting examples of Enterococcus selective mediumsinclude Slanetz-Bartley agar and Bile-esculine-azide agar.

[0043] Evidently and as will be further explained in the followingdescription, the screening method of the present invention involvedetermination of the relative fraction of a single type of bacteria orof several bacteria in order to determine the porbability of a subjectto have cancer. Each kind of bacteria being identified and isolated asdescribed above.

[0044] Referring again to FIG. 1, the screening method of the presentinvention may include the additional steps of (v) isolating one or moremicroorganisms from the sample for which their relative fraction wasdetermined (step 20); (vi) preparing a diagnostic sample containing oneor more of the isolated microorganisms, the fraction of themicroorganisms in the diagnostic sample corresponding to the relativefraction thereof in the fecal sample, as determined in step (iv) (step22) Dilution or re mixing of the microorganisms in a contolled manner asdefined above is essential in order to increase th effectiveness of themethod of the invention); and (vii) interacting (contacting) the mixturewith cancer cells for a time period sufficient to detect lysis of thecancer cells by the bacterial mixture (step 24), from which a tumor cellnecrosis index (TCNI) is determined for the specific tested fecal sample(steps 26, 28).

[0045] The cancer cells employed may be any standard culture of cancercells, for example MCF7. The interaction of the bacteria mixture withthe cancer cells includes incubation of the bacteria and cancer cellsunder conditions suitable for the bacteria to act on the cells. Theseconditions include suitable temperature (e,g. 37° C.), and a time period(in the following examples, 4 hours), sufficient determine the extent ofinteraction between the bacteria and the cancer cells, the extent ofinteraction is determined by the degree of lysis of the cancer cells bythe bacteria mixture (the diagnostic sample). This may be observed, forexample, by the aid of a microscope or an Automated Computer AssistedMicroscope, wherein the number of remaining cancer cells is counted.

[0046] FIGS 2A and 2B show microscope pictures obtained after incubationof cancer cells wih a bacterial sample obtained from a healthy subject,(FIG. 2A) or from a cancer patient (FIG. 2B) These picture show that inthe presence of a bacterial sample obtained from a sick subject only afew cancer cells remain, i.e. there is an effective lysis of the cancercells by the bacteria.

[0047] Referring again to FIG 1, the method of the invention maycomprise the steps of:

[0048] i) providing a fecal sample from said subject (step 10);

[0049] ii) treating said sample to obtain a feces-derived microorganismsample (step 12);

[0050] iii) identifying in the bacteria sample at least one type ofmicroorganism capable of expressing in a healthy subject L-asparaginaseII (L-PAR II) (step 14); and

[0051] iv) determining level of expression of L-PAR II or level ofactivity of L-PAR II (Step 30), the level is indicative of the presenceor absence of cancer cells in said subject (step 32).

[0052] L-asparaginase is an enzyme which catalyses the hydrolysis ofL-asparagine to aspartate and ammonia. It may be isolated from a numberof sources including bacteria, plants and animal tissues, but not fromhumans. Escherichia coli produces two forms of this enzyme, designatedtype I and type II. The two types of L-asparaginases differ in aserveral aspects, some of which are detailed below:

[0053] 1. L-aparaginase I (L-PAR I) is an enzyme located in thecytoplasm, whereas L-PAR II is a secreted protein.

[0054] 2. The activity of L-PAR II is regulated by oxygen levels in themedium such that a high level of induction is obtained under anaerobicconditions.

[0055] 3. L-PAR II has a much higher faffinity for is substrate thanL-PAR I.

[0056] 4. The two enzymes have different solubility and chromatographicbehavior as well as a different pH-dependent activity.

[0057] L-PAR II has been shown to inhibit the growth of a number ofanimal tumors in vivo, and to interfere with the synthesis of proteinsin vitro. The anti-tumor activiyt of L-PAR II may result from the needof the cancer cells for their rapid, malignant growth and thus survivala large amount of asparagines. These cancer cells use asparagines fromthe diet as well as from endogenously produced product (which islimited) to satisfy their large asparagines demand. Treatment of cancercells with L-PAR II was found to cause increased hydrolysis ofasparagine external to the cell, such that while normal cells are ableto provide themselves with all the asparagine they need internally,cancer cells become depleted rapidly and die.

[0058] As will be shown in the following examples, it has now been foundthat healthy individuals and cancer patients show striking differencesin their fecal microflora, and in particular in the levels of L-PAR IItherein which let to the claimed invention. In particular, the presentinvention disclosed the correlation between levels of L-PAAR II andcancer cells, such that low levels of epression of L-PAR II or ofactivity of L-PAR II, indicate the presence of cancer cells in saidsubject and vice versa.

[0059] As will be apprecaited by the skilled in the art, differentbacteria may epress L-PAR II. L-PAR II isolated from E. coli has beenextensively studied and appears as a tetramer of approximately 140 kDa.Thus, according to one preferred embodiment of the invention, thebacteria identified and isolated for detection of the level ofempression or activity of L-PAR II is E. coli.

[0060] The following description provides specific examples forexecuting the present invention. The present invention is defined by theclaims the contents of which are to be read as included within thedisclosure of the specification, and will be described in the followingnon-limiting examples. It should be clear that other variations in formand detail of the invention may be possible without departing from thescope and spirit of the invention as herein disclosed.

EXAMPLE I

[0061] Materials

[0062] Mac Conkey agar; m-Endo Agar Les; SInnetz-Bartley agar;Bile-esculine-azide agar; Simmons citrate; MIO medium Mueller Hintonagar; Esculine agar/TSA+6.5% NaCl (divided plate); and Indole teststrips were all purchased from Hylabs, Park Tamar, Rehovot, Israel.

[0063] Methods

[0064] Preparation of fecal samples

[0065] Samples of feces were homogenized, weigh (5 gr per sample) andintroduced each into sterile, 50 ml, tubes. Each sample was then dilutedwith sterile Saline (20 ml), vortexed for approximately 10-15 secondsand then incubated upright at room temperature for approximately 15minutes until large particles settle to the bottom of the tube.Alternatively, the samples may be spinned for 3 minutes at 400 rpm(Sorvall centrifuge) until the large particles settle at the bottom ofthe tube.

[0066] From each sample, the upper portion (i.e. not including the largeparticles) was removed and introduced into a second sterile tube furthervortexing for approximately 5 seconds. Alternatively, a bacteriologicalloop may be dipped into the upper portion of the sample for removal ofdesired aliquots. The resulting samples were then optionlly diluted withsterile Saline according to the following rations: 1.10², 1:10⁴, 1:10¹⁰,1:10¹².

[0067] Preparation of microorganism colonies

[0068] Aliquots (100 μl) from the 1:10² and 1:10⁴ diluted samples werehomogeneously inoculated on dry Slanetz-Bartley and Bile-esculine-azideplates (for isolation of Enterococcus sp).

[0069] In addition, aliquots (100 μl cach) from the 1:1010 and 1:1012dilutions were homogeneouly inoculated on dry MacConkey (for isolationof coliform containing Escherchia coli (E.coli)). Mueller Hinton agarplater we used to determine Gram negative and total bacteria count andwere inoculated in the same manner as with the MacConkey plates.

[0070] A sample fom the untreated (undiluted) feces (10 μl) was spreadon McConkey Agar plate or m-Endo Agar Les plates (the latter selectivefor coliform including E. coli. This “undiluted” sample is used toverify the “cleaning” and homogenization process does not changethefinal outcome in terms of types of bacteria and relative counting.

[0071] All plates were incubated for 12-24 hours at 37° C. untilcolonies of bacteria appeared on the plates. When Knecessary, typicallywith the Slantenz-Bartley and Bile-esculine-azide plates, incubation forup to 48 hours was performed.

[0072] All colonies grown on Mac-Conkey and Mueller Hinton plates werecounted for deteminaton of the total bacteria count.

[0073] Enterococcus sp. colonies appreaded on the Slantez-Bartley andBile-esculine azide plates as red or big black haloed coloniesrespectively. These Enterococus sp. colonies were counted. Thepercentage of the Entercococci colonies from the total number ofcolonies was determined.

[0074] Further, in m-endo-LES medium pink, off-white or blue-purpleColiform colonies appeared, being indicatve for the presence of E. colicolonies.

[0075]Streptococcus bovis colonies were determined as those beingBile-Esculine positive and Group D antigen positive, however. NaCl(6.5%) negative and PYR negative.

[0076] For the information of single colony plates, the followingprocedure was employed. By the use of a bacterialogical loop coliformcolonies were picked (quantity depends on quantitative analysis of thesample in order to maintain the orignal ratio) from each plate andtransfered onto a Mueller-Hinton Agar plat for incubation at 37° C.

[0077] Results

[0078] The presence or absence of Enterococcus sp. and/or Streptococcusbovis in the different plates was examined. Enterroccocus sp. bacteriawere identified as red colonies on Slanetz-Bartley or big black haloedcolonies on Bile-Esculine-azide as described above as well as thosecolonies that were Bile-Esculine positive and NaCl (6.5%) positive

[0079]Streptococcus bovis colonies were determined as those beingBile-Esculine positive and Group D antigen positive (enterococci bearthe Lancefield Group D antigen, however, NaCl (6.5%) negative and PYRnegative (Enterococcus produces to a positive PYR test (red colorpruduced additon of N, N methyl aminocynnamaldehyde reagent afterexposure to L-pyrrolidonyl-beta-naphtylamide (PYR) substrate).Streptococcus bovis was determined in the same manner.

[0080] The following Table 1 shows the results obtained. Positve resultsare indicate by “+” in the table. TABLE 1 Bile-Esculin NaCl (6.5%) PYRGroup D Antigen Enterococcus sp + + + + Streptococcus + − − + bovis

[0081] As indicated above, MacConkey Agar plates supports growth ofcoliform containing Enterobacteria (E.coil). Typically, E. coli apprearsas red colonies (lactose positive). In order to determine the fractionof E.Coli from the total Enterococci colonies and thereby from the totalbacteria count, the following tests were performed. (a) Indol test; (b)Simmons Citrate test; (c) Ornitine decarboxylase (ODC); and (d) motilitytest.

[0082]E.coli colonies were determined only when 98% of the bacteria wereIndol positive; 99% of the bacteria were Simmons Citrate negative; 65%of the bacteria were Ornitine decarboxylase (ODC) positive; and 95% ofthe bacteria were motile.

[0083] Alternatively, the indol, ODC and motility tests may be performedusing an MIO medium which enables conducting all three test in a singleprocedure.

[0084] The fraction of E.coli from the total coliform was determined bycalculating the pecentage of red colonies from the, total amount ofcolonies grown in the same Slanetz-Bartley agar plate or by calculatingthe percentage of red colonies on Slanetz-Bartley plates from the totalamount of colonies on Mueller-Hinton plates (more accurate). Theanalysis was performed using a spectrophotometer.

EXAMPLE II

[0085] Incubation of bacteria wih cancer cells

[0086] For evaluating the effect of the feces-derived microorganism oncancer cells, the following procedure was performed:

[0087] A supension of cancer cells (concentration of 4×104 cells/100μl), in serum-free DMEM was introduced into 96 well plates (6 duplicatesfor each stool sample).

[0088] Mueller-Hinton plates containing bacteria were washed with 10-mlDMEM and the bacterial suspension obtained was collected in 15-ml tubes.Aliquots of bacterial suspension (50 μl of bacterial suspension (2×10⁶bacteria/ml)) were then introduced into the tumor cell-containing wells.

[0089] For the formation of single colony plates, the procdure asdescribed above was employed. In particular, by the use of abacteriological loop coliform colonies were picked (quantity depends onquantitative analysis of the sample in order to maintain the originalratio) from each plate and transferred onto a Mueller-Hinton Agar platfor incubation at 37° C. to form the diagnostic sample for furtherincubation with the cancer cells.

[0090] Cancer cells-containing wells, in which no bacteria wereintroduced, were used as a regular negative control.

[0091] All wells were then incubated for 4 hours at 37° C. after whichthe content of each well was mixed and aliquots of the well content (130μl) were dried on glass slides at room temperature until the slidesbecame dry followed by fixation of the cells on the slide and stainingof the cells according to manufacturer instructions using the Giemsastain kit (Merck).

[0092] The extent of interaction between the test sample of bacteria andthe standard culture of cancer cells is made by counting the number ofcancer cells remaining in the visual field of the microscope, after anddyeing as necessary. The operative methods for counting cells on amicroscope slide are per se known to persons in the field and include,for example human manual counting or by the use of an Automated ComputerAssisted Microscope (ACAM) using a pre-defined algorithm (by counting 64fields per glass slide).

[0093] The automated Computer Assisted Microscope disclosed herein,performs the following principal steps:

[0094] 1) A three dimesional (3D) Auto-Focus is directed at the depth ofthe cells and not to the bacteria. Cells only from the slide window;

[0095] 2) By the use of a counting software, the cells are classified ashigh relevant, relevant, not relevant, highly not relevant cells, etc.,by analyzing each cell object to determine if its external membrane hasnot been disrupted (by the bacteria) or is “damaged”, i.e. at leastpartially lysed by the bacteria;

[0096] 3) Further, by the use of the software, cells are differentiaedfrom other objects of the same size and of a similar shapes (includingGimza dry stains);

[0097] 4) Yet further, by the use of the counting software, cells arecounted in a predefined window in order to minimize any deviations inthe resulting index as defined below.

[0098] A Tumor Cell Necrosis Index (TCNI) may be calculated which isindicative of the number of surviving cancer cells. The TCNI may bedetermined according to the following equation:

(a−b)/a×100=C

[0099] wherein

[0100] C=the tumor cell necrosis index (TCNI)

[0101] a=the number of cancer cells in the negative control samples(without the bacteria) or the number of standand cancer cells destroyedin the presence of the ‘control’ bacteria;

[0102] b=the number of cancer cells not destroyed by the tested sample.

[0103] Qualitatively, it will be apparent that in a healthy patient inwhich the intestinal bacteria have a similar activity to the ‘control’sample, be will be low giving a relatively high TCNI, whereas withcancer patients carrying cancerolytic cells b will have a relativelyhigh value and the TCNI will be lower.

[0104] Results

[0105]FIGS. 2A and 2B show microscope pictures of the result ofincubution of bacteria samples obtained from cancer patients or healthysubjects (diagnostic samples as prepared by the method of the presentinvention) with the standard cancer cells. In particular, a largeramount of viable cancer cells are visualized in the culture treated witha sample obtained from a healthy subject (FIG. 2A), as compared to theresult obtained with a bacteria sample obtained from a sick subject(FIG. 2B).

[0106] The results obtained were analyzed statistically based on a groupcontaining 110 subject of which 38 were active cancer patients, 36 werecancer patients with no evidence of diease (i.e. potentially cured, alsoreferred to as cancer NED subjects) and 36 were healthy subjects.

[0107] In the following analysis three cut-off values of TCNI weredetermined 50% cut-off, 60% cut off and 70% cut-off. For example, in thecase of 60% cut-off, if upon mixing the bacteria with cancer cells morethan 60% of the cells were destroyed then the test was regarded asnegative for cancer while if 60% or less of the cells were destroyed,the test was considered positive for cancer.

[0108] The following statistical results, were obtained (95% confidencelimits): 70% cut-off 60% cut-off 50% cut-off Sensitivity SensitivitySensitivity Active cancer 74% 68% 61% Non-cancer 61% 81% 86% Non-cancerSpecificity Specificity Specificity 61% 81% 86%

[0109] These results which had been obtain from General populationwithout selection show that the screening method of the presentinvention is sufficiently sensitive, i.e. capable of testing cancerpatients as positive for cancer as well as highly specific, i.e. capableof testing healthy subjects as negative for cancer).

[0110] In additon, for a chosen cut-off of 60% the Wilcoxon Rank SumTest was used in order to determine whether there is a statisicallysignificant shift in the index values between the cancer goups and thenon cancer groups. A rank-sum normal static with correction Z=−4.25, andp-value <0.0001 was obtained. This result shows that there is astatistically significant difference between the distribution of testscores in active cancer group and non cancer groups.

[0111] The effect of antibiotics on the validity on the index values wasalso evaluated. Out of the tested subjects, 1 of the 38 active subjectsand 10 of the non-cancer subject were treated with antibiotics. In theformer case an index value of 24 was obtained as compared to a meanvalue of 44.2 for the remainder of the group members, while in thelatter case a mean index value of 74.5 was obtained as compared to 66.9among the remainder of the group members.

[0112] These results may suggest that there is no apparent effect oftreatment with antibiotics on the TCNI obtained.

EXAMPLE III

[0113] Characterization of fecal bacteria derived L-PAR

[0114] In order to characterize L-PAR from fecal bacteria, theexpression levels of the enzyme by fecal derived bacteria wasdetermined. Assessment of the level of expression was performed byImmunoblots, using a specific antibody, and by assessing the amount ofspecific mRNA using conventional molecular methods such as reversetranscription (RT)-PCR. In addition, L-PAR II activity may be directlyevaluated by the use of a standard enzymatic assay.

[0115] Immunoblot

[0116] Fecal bacteria were grown anaerobically, collected and disruptedby sonication. Bacteria proteins were separated by polyacrylamide gelelectrophoresis, blotted to nitrocellulose and probed with asparainaseII-specific antiscra.

[0117] RT-PCR

[0118] Reverse transcription (RT)-PCR is a valuable tool widely used forgene expression. In bacteria, RT-PCR is helpful beyond standardprotocols of northern blot RNA/DNA hybridization to idenitify specifictranscripts. RT-PCR has been successfully used with differentmicoorganisms such as S. aureus, C. botulinum, M. tuberculosis andfungi.

[0119] Accordingly, in order to assess the transcription level of theenzyme bacteria is grown anaerobically fron which bacterial RNA isisolated quantative RT-PCR is performed using primers specific to L-PARII (according to published gene sequence of this enzyme). Ths Sequenceof the L-ASPARAGINASE II is well defined and may be available, interalia, through NCBI (Accession No. M34277).

[0120] It has been found that the presence of L-PAR II mRNA could bedetected without the need of amplification of the sequence by RT-PCR,while, in order to detect the mRNA from samples obtained from cancerpatients one or more RT-PCR amplifications were required. These resultsteach the correlation between the level of expression of L-PAR II andthe probability of a subject to have cancer, i.e. feces derived bacteriafrom healthy subjects have a high level of expression of L-PAR II whilefeces-derived bacteria from sick subject have a lower level ofexpression of L-PAR II.

[0121] L-Asparaginase Enzymatic Assay

[0122] Sheroplasts are generated by treating anaerobically grownbacteria with lysozyme and EDTA following ismotic shock. Under theseconditions, L-PAR II has been shown to be released into the medium whilemost of the L-PAR I remain intracellularly.

[0123] Asparaginase activity is assayed using the method of directNesslerization of ammonia according to which the rate of hydrolysis ofasparagine by the enzyme is determined. In particular, ammonia reactswiht Nessler's reagent to form a yellowish-brownish complex that can bequantified by spectrophotometer. Nessler reagent is an alkaline solutionof potassium merurie iodide commonly used in analytical chemistry,especially for testing the presence of ammonia in aqueous solutions(water sample blood sample or urine, sample).

[0124] Alternatively, it is possible to determine L-PAR II levels inmixtures of the two isoforms (I and II). The method requires calculatonof the ratio of activity at pII 5.0 and pH 8.4 and applying amathematical equation to correct the contribution of the activity ofeach isoform.

1. A method for an early diagnosis of cancer in a subject comprising thesteps: i) providing a fecal sample from said subject; ii) treating saidsample to obtain a feces-derived microorganism sample; iii) identifyingin the micoorganism sample one or more types of micoorganisms containedtherein; and iv) determining for each of said microorganisms itsrelative fraction from the total count of microorganisms in said sample,the relative fractions being indicative of the presence or abscence ofcancer in said subject.
 2. The method of claim 1, wherein said subjet isa human subject.
 3. The method of claim 1, wherein said microorganimsare isojated by colonies formation on seclective culture mediums.
 4. Themethod of claim 1, wherein said relative fraction of each of saidmicoorganisms is determined by calculating the percentage of saidmicroorganism from the total count of microorganisms in the same orcorresponding sample.
 5. The method of claim 1, wherein saidmicroorganism are bacteria.
 6. The method of claim 5, wherein saidbacteria are Gram-negative anaerobic bacteria.
 7. The method of claim 6,wherein said Gram-negative anaerobic bacteria is of a genus selectedfrom the group consisting of Escherichia, Salmonella, Shigella,Klebsiella, Yersinisa, Enterobacter, Hemophilus, Gandnerella andPasteurella
 8. The method of claim 7, wherein said bacteria is E.coli.9. The method of claim 8, wherein E. coli coliform is isolated from saidfecal sample by culturing the feces-derived sample of bactria on aculture medium selective for E.coli.
 10. The method nf claim 9, whereinthe culture medium is select from the group comsisting of McConkey agarand m-Endo agar
 11. The method of claim 5, wherein said bacteria areGram-positive bacteria.
 12. The method of claim 11, wherein saidGram-positive bacteria is of a genus selected from the group conistingof Staphylococcus, Enterococcus, Streptococcus, Lactococcus.
 13. Themethod of claim 12, wherein said bacteria is Streptococcus bovis and/orEnterocococcus sp.
 14. The method of claim 13, wherein Enterococcicoliform is isolated from said fecal sample by culturing thefeces-derived sample of bacteria on a culture medium selective forEnterococcus.
 15. The method of claim 14, wherein said culture medium isselected from the group consisting of Slanetz-Bartley agar andBile-esculined-azide agar.
 16. A method for an early diagnosis of cancerin a subject comprising the steps: i) providing a fecal sample from saidsubject; ii) treating said fecal sample to obtain a feces-derivedbacteria sample therefrom; iii) identifying in the bacteria sample oneor more types of micoorganisms; and iv) determining for each of saidmicoorganisms its relative fraction from the total count ofmicroorganisms in said sample or in a corresponding sample; v) isolatingone or more microorganisms from said sample for which their relativefraction was determined; vi) preparing a diagnostic sample containingone or more of the isolated micoorganisms, the fraction of themicroorganisms in said diagnostic sample corresponds to the relativefraction thereof in the fecal sample, as determined in step (iv); andvii) interacting said diagnostic sample with cancer cells for a timeperiod sufficient to detect lysis of said cancer cells by saiddiagnostic sample, thereby determining for said fecal sample a tumorcell necrosis index (TCNI).
 17. The method of claim 16, wherein saidfecal sample is a human fecal sample.
 18. The method of claim 11,wherein said treatment includes removal of undesired contamination fromsaid fecal sample to obtain an uncontaminated feces-derived bacteriasample.
 19. The method of claim 16, wherein said microorganisms areisolated by colonies formation on selective culture mediums.
 20. Themethod of claim 16, wherein said relative of each of said is determinedby calculating the percentage of said microorganism from the total countof micoorganisms the same bacteria sample.
 21. The method of claim 16,wherein said microorganisms are barteria.
 22. The method of claim 21,wherein said bacteria are Gram-negative anaerobic bacteria.
 23. Themethod of claim 22, wherein said Gram-negative anaerobic bacteria is ofa genus selected from the group consisting of Escherichia, Salmonella,Shigella; Klebsiella, Yersintsa, Enterobacter, Hemophilus, Gardnerellaand Pasteurella
 24. The method of claim 23, wherein said bacteria isE.coli
 25. The method of claim 24, wherein E.coli coliform is isolatedfrom said fecal sample by culturing the feces-derived sample of bacteriaon a culture medium selective for E.coli.
 26. The method of claim 25,wherein the culture medium is selected from the group consisting ofMacConkey agar and m-Endo agar.
 27. The method of claim 21, wherein saidbacterta are Gram-positive bacteria.
 28. The method of claim 27, whereinsaid Gram-positive bacteria is of a genus selected from the groupconsisting of Staphylococcus, Enterococcus, Streptococcus, Lactococcus.29. The method of claim 29, wherein said bacteria is Streptococcus bovisand/or Enterococcus sp.
 30. The method of claim 29, wherein Enterococcicoliform is isolated from said fecal sample by culturing thefeces-derived sample of bacteria on a culture medium selective forEnterococcus.
 31. The method of claim 30, wherein said culture, mediumis selceted from the group consisting of Slanetz-Bartley agar aiidBile-esculine-azide agar.
 32. The method of claim 16, wherein saidcancer cells are a standard culture of cancer cells.
 33. The method ofclaim 32, wherein said standard culture of cancer cells having theaccession No. ATCC MCF7.
 34. The method of claim 16, wherein saidmixture is interacted with the cancer cells for a time period sufficientto determine the extent of interaction between the bacteria and thecancer cells. 35, The method of claim 34, wherein the number ofinteracted and/or non-interacted cancer cells present at the end of saidtime period is determined, based on which a tumor cell necrosis index(TCNI) is calculated,
 36. A method for an early diagnosis of cancercomprising the steps: i) providing a fecal sample from said subject; ii)treating said sample to obtain a feces-derived micoorganism sample; iii)identifying in the micoorganism sample at least one type of micoorganismcapable of expressing in a healthy subject L-asparaginsae II (L-PAR II);and iv) determing level of expression of L-PAR II or level of activityof L-PAR II, said level is indicative of the presence or absence ofcancer cells in said subject.
 37. The method of claim 36, wherein saidfecal sample is a human fecal sample.
 38. The method of claim 36,wherein said treatment included removal of undesired contamination fromsaid fecal sample to obtain an uncontaminated feces-derived bacteriasample.
 39. The method of claim 36, wherein said micoorganisms areisolated from the feces-derived bacteria sample by colonies formation onselective culture plates.
 40. The method of claim 36, wherein saidmicoorganisms capable of expressing L-PAR II is E coil.
 41. The methodof claim 36, wherein low levels of expression of L-PAR II or of activityof L-PAR II, indicate the presence of cancer cells in said subject.